Structural determinants of miRNAs for RISC loading and slicer-independent unwinding

摘要

MicroRNAs (miRNAs) regulate expression of their target mRNAs through the RNA-induced silencing complex (RISC), whichcontains an Argonaute (Ago) family protein as a core component. In Drosophila melanogaster, miRNAs are generally sorted intoAgo1-containing RISC (Ago1-RISC). We established a native gel system that can biochemically dissect the Ago1-RISC assemblypathway. We found that miRNA-miRNA* duplexes are loaded into Ago1 as double-stranded RNAs in an ATP-dependent fashion.In contrast, unexpectedly, unwinding of miRNA-miRNA* duplexes is a passive process that does not require ATP or slicer activityof Agol. Central mismatches direct miRNA-miRNA* duplexes into pre-Ago1-RISC, whereas mismatches in the seed or guide strandpositions 12-15 promote conversion of pre-Ago1-RISC into mature Ago1 -RISC. Our findings show that unwinding of miRNAs is aprecise mirror-image process of target recognition, and both processes reflect the unique geometry of RNAs in Ago proteins.